Ribokinase from Leishmania donovani: purification, characterization and X-ray crystallographic analysis

Abstract

Leishmania is an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK from L. donovani was cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed the T_m to be 317.2 K. Kinetic parameters were obtained by functional characterization of L. donovani RK, and the K_m values for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space group P6₁, with unit-cell parameters a = b = 100.25, c = 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (V_M) of 2.45 ų Da^-1 and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

Keywords: Leishmania donovani; crystallization; d-ribose metabolism; ribokinase; thermal stability.

Figure 1

12% SDS–PAGE of purified recombinant LdRK after gel-filtration chromatography. Lane M, molecular-weight markers (labelled in kDa); lane 1, pooled purified fraction from gel-filtration chromatography.

Figure 2

(a) Far-UV circular dichroism spectrum of LdRK measured at 298 K in 7.5 mM Tris–HCl pH 8.5. (b) CD thermal denaturation curve of LdRK obtained by measuring the ellipticity at 222 nm in 1.0 K intervals from 293 to 343 K. (c) The first-derivative curve of thermal melting with a peak value (T _m) of 317.2 K.

Figure 3

Kinetic characterization of LdRK. (a) Plot of initial velocities against increasing concentrations of ribose. (b) Plot of initial velocities against increasing concentrations of ATP. Each data point represents the average from assays performed in triplicate; the standard error is shown as an error bar.

Figure 4

Crystallization of LdRK. (a) Crystals obtained with 0.1 M citric acid pH 4.3, 3.4 M NaCl, 4.5%(v/v) glycerol using the hanging-drop vapour-diffusion method. (b) Silver-stained SDS–PAGE of the crystals. Lane M, molecular-weight markers (labelled in kDa); lane 1, crystals of LdRK.

Figure 5

Diffraction image of LdRK at 1.95 Å resolution obtained using a MAR 225 CCD detector.